PROJECT SUMMARY During autoimmune disease, the body incorrectly identifies ?self? molecules as foreign and mounts a chronic immune attack. Conventional therapies employ broad immunosuppression, which has provided significant benefits to patients, but can leave these individuals immunocompromised. This limitation, along with the lack of cures for most autoimmune diseases, has sparked intense interest in strategies that could control autoimmunity with vaccine-like specificity, leaving the rest of the immune system intact. Several pre-clinical reports and clinical trials have investigated this theory to combat multiple sclerosis (MS), a neurodegenerative disease in which myelin in the central nervous system (CNS) is attacked by the immune system. An important finding from these studies is that co-administration of myelin peptide and tolerizing immune signals can promote the development of regulatory T cells (TREGS) that ameliorate disease. The polarization of nave T cells into inflammatory T cells (e.g., TH17) or TREGS is localized to lymph nodes (LNs), the tissues that coordinate adaptive immunity. However, the link between the combinations, concentrations and persistence of immune cues in LNs, and the extent and specificity of systemic tolerance elicited, is not well understood. New knowledge of how signal integration in LNs drives tolerance could help address limitations associated with current therapies, such as incomplete control of disease and non-specific suppression. This proposal will study these fundamental questions in disease using a new platform that combines direct intra-LN (i.LN.) injection with controlled release biomaterial depots. Preliminary data in mice demonstrate that a single dose of depots co-encapsulating two of the most studied signals ? myelin peptide and rapamycin, a drug known to promote TREGS ? permanently reverses disease-induced paralysis in a model of MS (EAE). These effects occur even when depots are administered at the peak of disease, confirming the power of this system to serve as a tool to locally control the function of one LN, while dissecting the impact on systemic tolerance and at distant sites such as the CNS, spleen, and distal LNs. We hypothesize that this platform will allow previously inaccessible questions to be addressed, including the roles that local signals, combinations, and kinetics within LNs play in programming the nature of tolerance. The specific aims are 1) determine how local signals in LNs polarize T cell function and program systemic tolerance, 2) decipher the impact of signal location, delivery route, and kinetics on T cell polarization, 3) compare the local structure and function of depot-treated LNs to distal LNs, spleen, and CNS, and 4) test if the link between local function and systemic tolerance is generalizable to other self-antigens. This work will generate insight that informs design of new therapies that aim to promote tolerogenic function in an antigen-specific manner during autoimmune diseases such as MS, Type 1 diabetes, and rheumatoid arthritis.